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Indian Journal of Pharmacology, Vol. 41, No. 1, January-February, 2009, pp. 36-40 Research Article Evaluation of hepatoprotective activity of Cleome viscosa Linn. extract Gupta NishantKumar, Dixit VinodKumar Department of Pharmaceutical Sciences, Dr. H. S. Gour Vishwavidyalaya, Sagar, MP Date of Submission: 11-Jul-2007 Code Number: ph09009 Abstract Objectives: To evaluate the hepatoprotective activity of ethanolic extract of Cleome viscosa Linn. (Capparidaceae) against carbon tetrachloride (CCI 4 ) induced hepatotoxicity in experimental animal models.Materials and Methods: Leaf powder of Cleome viscosa was extracted with ethanol. The hepatoprotective activity of the extract was assessed in CCI 4 induced hepatotoxicity in rats. Various biochemical parameters were estimated and histopathological studies were also performed on rat liver. The hepatoprotective activity was also supported by determining a functional parameter, i.e. thiopental-induced sleep of mice poisoned with CCl 4 . Results: The test material was found effective as hepatoprotective, through in vivo and histopathological studies. The extract was found to be effective in shortening the thiopental induced sleep in mice poisoned with CCl 4 . The hepatoprotective effect of ethanolic extract was comparable to that of silymarin, a standard hepatoprotective agent. Conclusion: The results of the present study show that ethanolic extract of Cleome viscosa has significant hepatoprotective activity. Keywords: Carbon tetrachloride; Cleome viscosa Linn; hepatoprotective Introduction Cleome viscosa Linn. (Capparidaceae) is a widely distributed herb with yellow flowers and long slender pods containing seeds. The whole plant is sticky in nature and has a strong odour resembling asafoetida. It is found throughout the greater part of India, often in waste places and is known as Hurhur (Hindi) in Indian traditional medicine. Traditionally, this plant is used in various disorders such as diarrhoea, fever, inflammation, liver diseases, bronchitis, skin diseases, and malarial fever. The juice is useful in piles, lumbago and earache. [1] The analgesic, antipyretic and anti-diarrhoeal activities of the extract have been reported by researchers. [2],[3],[4] In our field studies of ethno-medicinal plants of Bundelkhand, it was noted that the fresh leaves of Cleome viscosa are widely used as medicine for jaundice. A systematic study to assess the action of the plant for liver diseases has not been reported and hence the present investigations were undertaken.Materials and Methods Plant material Chemicals Animals Preparation of extract 10 g ethanolic extract was dispersed in 200 ml of distilled water and extracted with ethyl acetate (200 ml) in a separating funnel. Ethyl acetate fraction was separated and concentrated under reduced pressure and low temperature (40-50°C) in a rotary evaporator (Yield 0.732 % w/w). This part of the test sample was named fraction A. Carbon tetrachloride induced hepatotoxicity model Assessment of hepatoprotective activity The hepatoprotective activity was confirmed through histopathological studies on liver of rats. After collection of blood for biochemical estimation, the rats were sacrificed and the liver was carefully dissected, cleaned of extraneous tissue, and fixed in 10% formalin for at least 24 h. Then the paraffin sections were prepared (automatic tissue processor, Autotechnique) and cut into 5 µm thick sections, using a rotary microtome . The sections were stained with Haematoxylin-Eosin dye and studied for histopathological changes. [9] Barbiturate sleeping time The time of lost reflex induced by short acting barbiturate (thiopental sodium) is significantly prolonged in the event of hepatic damage and can be used as a measure of the functional status of the drug metabolizing enzymes in the liver. [10],[11],[12] Thirty-six Swiss mice were selected and randomly divided into six groups of six each. Group A was kept as normal and normal sleeping time was determined after injecting the thiopental sodium (25 mg/kg b.w.). Group B was kept as control and administered CCl 4 (1 ml/kg b.w.). Group C and D were treated with ethanolic extract 100 mg/kg b.w. and 200 mg/kg b.w. respectively. Group E and F were given a dose of fraction A (10 mg/kg b.w.) and silymarin (50 mg/kg b.w.) respectively. After 24 h, a dose of CCl 4 (1 ml/kg b.w.) was given to B, C, D, E and F groups. Sleeping time was determined against thiopental sodium injection (25 mg/kg b.w.) after two hours of CCl 4 injection. CCl 4 was administered intra-peritoneally, whereas the extracts, fraction and silymarin were given orally. Statistical analysis Results In vivo hepatoprotective activity Barbiturate sleeping time Histopathological studies In the liver section of the animals treated with ethanolic extract (100 mg/kg b.w.) + CCl 4 , the nucleii are not very clear as in normal hepatocytes; however, when compared to the CCl 4 damaged group, the number of hepatocytes with normal nucleus was much more. The endothelium is disrupted in places. Pyknotic nucleus and vacuolation in cytoplasm are observed to be low, as compared to the CCl 4 group [Figure 3a]. The hepatic cells of rats treated with ethanolic extract (200 mg/kg b.w.) and intoxicated with CCl 4 were radially arranged. The vacuolation was present, similar to that of normal. The recovery was comparable to that with silymarin, a standard hepatoprotective agent. The intralobular vein was normal in structure, but the wall was damaged, as shown in [Figure 3b]. No pyknosis in the nucleus was seen. Liver sections of rats that were treated with fraction A along with CCl 4 exhibited a high degree of damage suggesting negligible protection to the liver by fraction A. Discussion The presence of jaundice is a cardinal feature of liver disease and its presence usually shows disturbance involving the hepatobiliary system. [14] Carbon tetrachloride is a potent hepatotoxin, and a single exposure to it can rapidly lead to an increase in the level of several enzymes, severe centrizonal necrosis and steatosis. [15] The hepatotoxicity induced by CCl 4 is due to its metabolite CCl 3·, a free radical which binds to lipoprotein and leads to peroxidation of lipids of the endoplasmic reticulum. [16] The ability of a hepatoprotective drug to reduce the injurious effects or to preserve the normal hepatic physiological mechanisms, which have been disturbed by a hepatotoxin, is the index of its protective effects. The lowering of enzyme level is a definite indication of the hepatoprotective action of the drug. Protection of hepatic damage caused by carbon tetrachloride administration was observed by recording SGOT, SGPT, SALP and SBLN levels in different groups. [17] The transport function of the hepatocytes is disturbed in hepatic injury, causing the leakage of enzymes due to altered membrane permeability. [18] Although both the doses of ethanolic extract offer hepatoprotection, the higher dose (200 mg/kg b.w.) is more effective [Table - 1] and [Table 2]. Depending on the type of cell and the membrane involved, lipoperoxidation due to CCl 4 results in hemolysis, which increases the serum bilirubin level. [16] The 200 mg/kg dose of extract showed an effective derange in the elevated serum bilirubin level, as compared to the low dose. The extract thus has a potential application in the acute condition of jaundice. A possible mechanism of the extract on bilirubin levels may be interference with cytochrome P 450 , resulting in the hindrance of the formation of hepatotoxic free radicals, thereby protecting the integrity of the membrane. The ability of the ethanolic extract of Cleome viscosa Linn. to reduce the prolongation of thiopental-induced sleep in CCl 4 poisoned rats is further indicative of the hepatoprotective potential of the test substance. It has been established that since the barbiturates are metabolized almost exclusively in the liver, the sleeping time after a given dose is a measure of hepatic metabolism. [12] If there is any liver damage, as in this case by CCl 4 intoxication, the sleeping time after a given dose of the barbiturate will be prolonged, because the amount of the barbiturate metabolized per unit time will be less. The histopathological studies also exhibit the efficacy of drug as a hepatoprotective. Simultaneous treatment of ethanolic extract with CCl 4 produces lesser degree of damage to the liver cells as compared to the animals treated with CCl 4 alone. The sections of the liver treated with extract (200 mg/kg b.w.) and CCl 4 reveal better hepatoprotective activity almost similar to the standard (silymarin) group. Negligible damage to a few hepatocytes present in the close vicinity of the intralobular vein is observed. Hepatocytes show normal appearance with the presence of vacuoles in the cytoplasm. These data, along with the histopathological studies, clearly show the hepatoprotective activity of Cleome viscosa Linn. and justify the use of this plant for jaundice.[Figure 3c],[Figure - 4] Acknowledgments The authors would like to express their deepest thanks to Micro Labs Limited, Goa, India for providing a gift sample of silymarin. The authors would also like to acknowledge National Botanical Research Institute, Lucknow, India for providing the facility for the in vivo study. The award of junior research fellowship (UGC) to one of the authors (N. K. Gupta) for undertaking these studies is thankfully acknowledged.References
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